This competing renewal application seeks continued support for a primary research in the Pi's laboratory using the computational approaches to elucidating the molecular mechanisms of general anesthesia. Research in the previous funding period challenged the traditional structure-function paradigm in explaining the action of general anesthetics on ion channel proteins (Tang&Xu, PNAS, 99:16035-16040, 2002) and proposed an alternative viewpoint that the effects of general anesthetics on protein global dynamics on the timescale matching the characteristic time of protein function might underlie a common mechanism of action of general anesthetics. To test this central hypothesis, we will take 4x4 approach to integrate 4 complementary state- of-the-art computational methods with 4 levels of validation with experimental data. We will focus on the anesthetic-hypersensitive neuronal (04)2(32)3 nicotinic acetylcholine receptor (nAChR) and the anesthetic- insensitive (a7)5 nAChR, as well as the Torpedo isoform of the muscle-type (alkplvQ nAChR. A novel integration of homology modeling, molecular dynamics (MD) simulations in a fully hydrated ternary membrane patch, coarse-grained normal mode analysis (NMA), and Brownian dynamics (BD) will be used to generate and validate high-resolution, closed and putatively open structural models for (alkpIvS, (a4)2(p2)3 and (a7)5 nAChR on the basis of the 4-A resolution structure of the (a1)2p1v5 nAChR as a template. Flexible ligand docking or manual docking of anesthetics (halothane and isoflurane) at experimentally identified anesthetic-binding sites will be followed by MD equilibration to encode anesthetic effects on tertiary and quaternary structures. NMA and BD will then be used to quantify the gating-related low-frequency motions of the receptors and ion permeation across the channel. Two groups of mutations that changed nAChR's sensitivity to anesthetics experimentally will be tested for global dynamics changes. Four levels of experimental validation for structures will include agonist- binding affinity, topology matching to low-resolution experimental structures and pore residue accessibilities, I-V curve calculations, and cation/anion and mono-/di-valence ion permeability ratios. Our substantive amount of preliminary results supports the following four specific aims: (1) To generate and validate, using existing experimental data, the closed- and putative open-channel structures of the neuronal (a4)2(p2)3 nAChR (hypersensitive to volatile anesthetics) and (a7)5 nAChR (insensitive to volatile anesthetics) as well as the open- channel structure for Torpedo (al^plvfi nAChR (sensitive to volatile anesthetics); (2) To perform extensive, multi-seed MD simulations on wild type and mutant channels in the absence and presence of anesthetics; (3) To carry out normal mode analysis to determine anesthetic effects on global dynamics, using the fully equilibrated structures in SA#2 as input; and (4) To relate anesthetic effects on global dynamics to channel function by performing Brownian dynamics calculations of ion permeation through the putative open-channels. The research will bridge the experimental and theoretical understanding of anesthetic effects on channel proteins, thereby facilitating the future design of new and novel anesthetic drugs that are more specific with fewer side effects.